Modified avidin and streptavidin and methods of use thereof

ABSTRACT

The present invention provides modified avidin and streptavidin compounds that have suitable blood clearance kinetics for use in a two-step approach to deliver a molecule to a target site. In particular, to hasten SA&#39;s blood clearance carbohydrate moieties are covalently bonded to SA. To prolong Avid&#39;s blood clearance, Avid is deglycosylated and/or neutralized by alkylation of its lysine amino acids. In a two-step approach, biotinylated compounds are used to deliver radionuclides, cytotoxic drugs, MRI agents, fluorochromes and other agents suitable for imaging and therapy to target-bound modified streptavidin or avidin conjugated antibodies or other targeting agents.

FIELD OF THE INVENTION

The present invention provides modified avidin and streptavidincompounds that have optimal blood clearance kinetics for use in atwo-step approach to deliver a molecule to a target site. In particular,to hasten streptavidin's blood clearance, carbohydrate moieties arecovalently bonded to streptavidin. To prolong avidin's blood clearance,avidin is deglycosylated and/or neutralized by alkylation of its lysineamino acids. The present invention further provides targeting agents,such as monoclonal antibodies, conjugated to modified streptavidin andavidin. In a two-step method of imaging or therapy, biotin conjugatesare used to deliver radionuclides, cytotoxic drugs, magnetic resonanceimaging agents, fluorochromes and other agents suitable for imaging andtherapy to target-bound conjugates of modified streptavidin or avidinand antibodies or other targeting agents.

BACKGROUND OF THE INVENTION

Various diagnostic, therapeutic, fluorescent and enzyme linkedapplications utilize cell or tissue specific targeting agents asdelivery systems for radioactive, paramagnetic, cytotoxic or therapeuticagents. Any agent which is specific for a lesion or site of interest canpotentially act as a targeting agent. For example, polyclonal andmonoclonal antibodies can be produced which exhibit considerablespecificity for certain cell or tissue types. Many other agents,including toxins such as diphtheria toxin, exhibit cell specificity andcan be used to deliver diagnostic or therapeutic agents. The techniqueof delivery of monoclonal antibodies (MAbs) has been investigated forcancer therapy as well as for diagnosis of cancer, thromboembolism andcardiac myopathy.

For successful imaging with directly labeled antibodies, sufficientlabeled MAb must localize at the target site to provide enough signalfor detection. Target-to-background ratios must be high in order toachieve adequate contrast between target-bound radioactivity andbackground levels in other organs, tissues and blood. A major obstacleto successful imaging with directly labeled antibodies is the highbackground activity of free circulating radiolabeled MAbs due toprolonged circulation and accumulation in liver and spleen, the normalmetabolic sites for Abs. Furthermore, the toxic effects of highradiation doses must be considered in both radioimmunotherapy andradioimmunoimaging. Such obstacles are also a consideration for methodsutilizing targeting agents other than monoclonal antibodies.

Galactose protein modification has been utilized in an attempt tomanipulate clearance of antibodies. For example, Ong et al. (1991)Cancer Res. 51:1619 and Mattes (1987) JNCI 79:855 have conjugatedgalactose to radiolabeled MAbs to manipulate the blood clearance rate ofthe MAbs in both diagnostic and therapeutic techniques. U.S. Pat. No.4,401,647 discloses the galactose modification of albumin for liverimaging. Vera et al. (1985) J. Nucl. Med. 26:1157 and Stadalnik et al.(1992) Investig. Radiol. 28:64 have conjugated galactose to albumin forfunctional imaging studies of the liver. Both of these procedures resultin radionuclide accumulation in the liver. This is problematic forimaging liver or chest lesions. In addition, liver metabolism increases,which creates problems for imaging and therapy due to accumulation ofradioactivity in radiosensitive organs and tissues, especially bonemarrow.

To overcome such obstacles, "pre-targeting" approaches have beeninvestigated. See, e.g., Hnatowich, et al (1987) J. Nucl. Med. 28: 1294.In the conventional one-step method the radionuclide is linked to theMAb either directly or via a bifunctional chelating agent. In thepre-targeting approach the antibody is unlabeled, but conjugated to abinding moiety such as avidin or streptavidin. Unlabeled antibody isadministered, and antibody which does not localize to the target site isallowed to clear from circulation or removed by a clearing agent beforethe administration of radioactivity. The radioactivity is thenadministered in a chemical form which has high affinity for theantibody, e.g., bound or chelated to the binding partner of the moietyconjugated to the antibody.

To provide the diagnostic or imaging agent in a form with high affinityfor the antibody, two-step methods have been designed to exploit thehigh affinity of avidin and streptavidin for biotin. Avidin, a 67kilodalton (kD) glycoprotein found in egg whites, has an exceptionallyhigh binding affinity (K_(d) =10⁻¹⁵ M) for biotin. Avidin consists offour subunits, each capable of binding one biotin molecule.Streptavidin, a similar protein produced in Streptomyces avidinii,shares significant conformation and amino acid composition with avidin,as well as high affinity and stability for biotin. However, streptavidinis not glycosylated and reportedly exhibits less non-specific binding totissues. Streptavidin is widely used in place of avidin because of itslower non-specific binding. Biotin, a member of the B-complex vitamins,is essential for amino acid and odd-chain fatty acid degradation,gluconeogenesis and fatty acid synthesis and is normally found in theenzyme bound form as biocytin.

In the prior art approach to radioimaging or radiotherapy, antibodiesare coupled with either biotin or streptavidin and administered to thesubject, followed by administration of radiolabeled streptavidin orbiotin, respectively. Hnatowich et al. (1987); Kalofonos et al. (1990)J. Nucl. Med. 31:1791; Paganelli et al. (1992) Eur. J. Nucl. Med.19:322; Yao et al (1995) J. Nucl. Med. 36:83. A three-step approach,which involves the administration of biotinylated antibody, followed bystreptavidin and then radiolabeled biotin, has also been investigated.Paganelli et al. (1988) Int. J. Cancer 2:121. These multistep proceduresgenerally require large doses of protein, and long time durations, oftendays, to complete. In particular, the prolonged circulation ofstreptavidin-conjugated antibodies requires either a significant timeinterval to allow clearing or the use of a clearing agent beforeadministration of radiolabeled streptavidin or biotin. Paganelli et al.(1992) Eur. J. Nucl. Med. 19:322; U.S. Pat. No. 4,863,713 to Goodwin etal. Conversely, lavidin conjugated moieties would be expected to cleartoo quickly, and would likely not be useful in pre-targeting approachesdue to unacceptably low target accumulation.

Accordingly, the pre-targeting method of the prior art is a complicatedsystem that suffers from practical limitations, including, for example,the pharmacokinetics of avidin and streptavidin. Streptavidin and avidinexhibit markedly different pharmacokinetics after intravenous injection,with avidin clearing from the blood much faster than streptavidin. Thus,for a rapid two step procedure, streptavidin-containing moieties clearslowly, necessitating a delay in the injection of radiobiotin untilblood levels have decreased. Conversely avidin clears too quickly andaccumulates in the liver and kidney, thus resulting in low targetaccumulation. Avidin's rapid blood clearance results from its inherentpositive charge and its mannose terminal sugars which may bind tomannose receptors present in the liver on Kupffer cells.

Thus, the directly labeled antibody approaches of the prior art sufferfrom background dosimetry problems, and the pretargeting approaches ofthe prior art are complicated, requiring large doses, multiple steps,and significant amounts of time to perform.

The present invention overcomes the deficiencies of the prior art. Inparticular, the present invention provides modified avidin andstreptavidin compounds that have optimal blood clearance kinetics foruse in a rapid method for delivery of an agent to a target site.Further, the avidin and streptavidin compounds of the invention are notradiolabeled, thus avoiding interference from background activity.Because of the optimal clearance kinetics of the present compounds, anyof the subsequently administered radiolabeled biotin that does notbecome target bound is quickly filtered and removed from the body. Thepresent invention thus satisfies the prior art need for a rapid andefficient method of imaging and therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents isoelectric focusing of streptavidin (SA) andstreptavidin-galactose (SA-gal) samples. The ordinate represents theisoelectric point and the abscissa represents molar incubation ratios ofgalactose.

FIG. 2A represents isoelectric focusing of Avid and Avid-N.

FIG. 2B represents sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) of Avid and Avid-N.

FIG. 3 diagrams the synthesis of Tgal-SA.

FIGS. 4A and 4B represent a deferoxamineacetyl cysteinyl biotin (DACB)saturation assay on SA, SA-gal, avidin (Avid) and neutralized avidin(Avid-N). The abscissa indicates molar incubation ratios of galactose oracetic acid N-hydroxysuccinimide ester (NHS-Ac) with protein. Theordinate indicates bound DACB/protein ratios.

FIG. 5A is a graph showing blood pharmacokinetics of SA and SA-gal inrabbits. The abscissa represents time and the ordinate representspercentages of the injected dose of compound.

FIG. 5B represents the biodistribution of ¹³¹ I-SA and ¹³¹ I-SA-gal at 2hours after injection in the blood, spleen, kidneys, liver and lung.

FIG. 6 represents nuclear images of ¹³¹ I-SA and ¹³¹ I-SA-gal at 5, 30and 120 minutes after injection.

FIG. 7A is a graph showing blood pharmacokinetics of ¹³¹ I-Avid, ¹³¹I-deglycosylated avidin (Avid-E), ¹³¹ I-Avid-N, ¹³¹ I-deglycosylated andneutralized avidin (Avid-E/N) in rabbits.

FIG. 7B represents the biodistribution of ¹³¹ I-Avid, ¹³¹ I-Avid-E, ¹³¹I-Avid-N and ¹³¹ I-Avid-E/N at 2 hours after injection in the blood,spleen, kidneys, liver and lung.

FIG. 8 represents nuclear images of ¹³¹ I-Avid, ¹³¹ I-Avid-E, ¹³¹I-Avid-N and ¹³¹ I-Avid-E/N at 5, 30 and 120 minutes after injection.

FIG. 9 is a graph showing blood pharmacokinetics of ¹²⁵ I-SA and ¹³¹I-streptavidin-trigalactose (SA-Tgal) in rabbits. The abscissarepresents time and the ordinate represents percentages of the injecteddose of compound.

FIG. 10 is a composite of the nuclear images of ¹³¹ I-gal-SA at 5, 30and 120 minutes.

FIG. 11 is an outline of the synthesis of GC4-SA, GC4-SA-gal andGC4-A-Tgal.

FIG. 12 represents SDS-PAGE analysis for the conjugation andpurification of SA-GC4.

FIG. 13A is a graph representing the bound ratio of PDP/SA compared tothe N-succinimidyl-3- 2-pyridyldithio!propionate (SPDP/SA) incubationratio at pH 6.5 and 8.0.

FIG. 13B represents isoelectric focusing analysis on SPDP/SA sampleswith different incubation ratios at pH 6.5 and 8.0.

FIG. 14A is a graph representing a comparison of the blood clearancerates of ¹²⁵ -SA-GC4, ¹³¹ I-gal-SA-GC4 and ¹³¹ I-SA-Tgal-GC4.

FIG. 14B represents the biodistribution of ¹²⁵ I-SA-GC4, ¹³¹I-gal-SA-GC4 and ¹³¹ -SA-Tgal-GC4.

FIG. 15 is a composite of the nuclear images of ¹³¹ I-gal-SA-GC4 and ¹³¹I-Tgal-SA-GC4 at 5, 30 and 120 minutes.

SUMMARY OF THE INVENTION

The present invention provides modified avidin (Avid) and streptavidin(SA) compounds that have blood clearance kinetics suitable for use indiagnostic and therapeutic methods. In particular, to hastenstreptavidin's blood clearance carbohydrate moieties are covalentlybonded to streptavidin. To prolong avidin's blood clearance, avidin isdeglycosylated and/or neutralized by alkylation of its lysine aminoacids.

The present invention further provides conjugates of modified avid or SAand a targeting agent. In a preferred embodiment the targeting agent isan antibody or antibody fragment.

A further aspect of the present invention provides a method to deliver adiagnostic or therapeutic agent to a target site. The method of deliveryof a diagnostic agent comprises administering to a host a modified Avidor SA conjugated to a targeting agent in an amount sufficient to bind toa target site, followed by administering a detectable biotinylatedcompound under conditions to form a complex with the Avid or SAconjugated targeting agent and at a dose sufficient for detection. Theresulting complex is then detected. In a preferred embodiment thetargeting agent is an antibody or antibody fragment. In a preferredembodiment, the present method utilizes fibrin specific MAbs for thediagnosis of intravascular lesions.

The method of delivery of a therapeutic agent comprises administering toa host a modified Avid or SA conjugated to a target agent, followed byadministering a therapeutic biotinylated compound under conditionssufficient to form a complex with the Avid or SA conjugated targetingagent and at a therapeutically effective dose. In a preferred embodimentthe targeting agent is an antibody or antibody fragment.

Yet another aspect of the present invention provides pharmaceuticalcompositions containing the subject conjugates and a pharmaceuticallyacceptable carrier. A compartmentalized kit for imaging or therapy isalso provided by the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides modified avidin (Avid) and streptavidin(SA) compounds that have blood clearance kinetics suitable for use in atwo step approach to deliver a molecule to a target site. The presentcompounds are useful in a method of delivering a diagnostic ortherapeutic agent to a target site. In the present method, the modifiedAvid or SA is conjugated to a targeting agent and delivered to a host inan amount and under conditions to bind to a target site. A detectable ortherapeutically effective biotinylated compound is then administeredunder conditions to form a complex with the modified Avid or SAconjugated targeting agent and at a dose sufficient for detection ortherapeutic efficacy.

Modified Avid and SA compounds having suitable blood clearance kineticsfor two step imaging or therapy are defined herein as compounds that,when conjugated to a targeting agent, circulate long enough toaccumulate at a target site in sufficient amounts to bind a detectableor therapeutically effective amount of a biotinylated compound andfurther that are sufficiently cleared from circulation before thesubsequent administration of biotin. Sufficient clearance of circulatingmodified Avid or SA conjugates is that amount of clearance that preventsclinically or diagnostically unacceptable levels of background due tobinding of a subsequently administered biotinylated compound to thecirculating modified Avid or SA conjugates to a biotinylated compound. Asubsequently administered biotinylated compound is one that isadministered within 24 hours of administration of the modified Avid orSA conjugate. In a preferred embodiment the biotinylated compound isadministered within six hours, or more preferably within one hour, ofthe administration of the Avid or SA conjugate.

The modified SA compounds of the present invention are cleared morerapidly from the circulation than unmodified SA. In particular, thepresent invention provides SA covalently modified to contain at leastone carbohydrate moiety. While not intended to limit the presentinvention, it is likely that enhanced clearance of the modified SAcompounds results from binding to the liver as galactose receptors.Because the SA compounds are internalized by the galactose receptor,there is a reduced potential of immunogenicity.

The modified SA compounds are prepared by covalently attaching acarbohydrate moiety through at least one of SA's twenty amino groups.The resulting compound may contain linker molecules that facilitate theconjugation of carbohydrate to SA. From one to twenty of SA's aminogroups (of which sixteen are lysine amino residues and four are terminalamino residues) may be modified by conjugation to carbohydrate. Further,more than one carbohydrate unit may be bonded per SA amino group, forexample by providing branched compounds. Preferred carbohydrate moietiesare galactose, mannose, fructose and lactose. Galactose is particularlypreferred. In a most preferred embodiment, trigalactose moieties arecovalently bonded to SA through SA's amino groups.

The modified SA compounds of the present invention contain at least onecarbohydrate moiety. The carbohydrate moiety is preferably covalentlybonded through an amino group of SA. The carbohydrate moiety ispreferably galactose. The galactose moiety may consist of more than onegalactose unit. Trigalactose is particularly contemplated. In apreferred embodiment, from about 1 to about 20 of SA's amino groups arecovalently bonded to galactose.

The carbohydrate-modified SA compounds of the present invention can beprepared by synthetic methods know in the art. Either or both of SA andcarbohydrate may be derivatized with a reactive group to facilitatecovalent bonding. For example, galactose can be covalently attached toSA via a nucleophilic reaction of SA's amino groups with a reactivegalactose-containing species such asα-D-galactopyranosyl-phenylisothiolcyanate.

In a general procedure, α-D-galactopyranosyl-phenylisothiocyanate issolubilized in methanol and added to SA solubilized in 0.05M sodiumphosphate/0.15 NaCl buffer. α-D-galactopyranosyl-phenylisothiocyanate isadded to SA in molar ratios of 10, 25 and 50. The preferred galactose/SAincubation ratio is about 25 to 50. A ratio of 50 results in nearsaturation of SA's amino groups while maintaining adequate bindingaffinity for biotin. The resulting solution is then incubated overnightat room temperature and the resulting SA-gal product separated, forexample, by ultrafiltration.

The resulting SA-gal product contains galactose moieties covalentlybound to SA. This product can then be analyzed to determine the amountof galactose bonded per SA molecule. For example, the average boundratio of galactose to SA can be determined indirectly by quantifyinggalactose concentrations in the ultrafiltrate by an anthronecalorimetric assay, a method known to one of ordinary skill in the artand described by Roe (1955) J. Biol. Chem. 212:335.

Modified SA compounds in which more than one carbohydrate moiety iscovalently bonded through each SA amino group can also be synthesized bymethods known in the art. For example, cluster glycosides suitable forattachment to proteins are known in the art. Lee (1978) CarbohydrateResearch 67:509 disclose a trigalactose derivative,(6-aminohexamido)tris(B-O galactosyloxymethyl) methane (Tgal) containingthree galactose moieties at one end and a primary amine at the other.Trigalactose is particularly preferred for SA modification in accordancewith the present invention since synthetic triglycosides exhibit higherbinding affinity to the galactose receptor compared to the correspondingmonoglycosides. Connolly et al (1982) J. Biol. Chem. 257:939.

The present invention provides additional trigalactose derivativessuitable for coupling to proteins. The trigalactose derivatives arestable and conveniently prepared. In one embodiment, a sulfhydryl groupis incorporated into Tgal by reacting Tgal with S-acetylthioacetic acid(SATA) to provide Tgal-ATA. For example, a 3/1 molar ratio of SATA indimethylsulfoxide is added to Tgal in 0.1M PO₄ at pH 8.0 and incubatedovernight at room temperature under N₂. Tgal-ATA can then be purified,for example, by G-10 SEPHADEX size exclusion chromatography. Tgal-ATAhas the formula: ##STR1## wherein gal is ##STR2##

In another embodiment, a sulfhydryl group is incorporated into Tgal byreacting 2-iminothiolane (Traut's reagent) to provide Tgal-SH. Forexample, 2-iminothiolane is added in two equal aliquots to Tgal for afinal molar ratio of 20/1 in 0.1M PO₄, 1 mM EDTA pH 7.6 and stirred forone hour at room temperature under N₂. Tgal-SH can then be purified, forexample, by G-10 SEPHADEX size exclusion chromatography. Tgal-SH has theformula: ##STR3##

The trigalactose derivatives are utilized to covalently attachtrigalactose moieties to SA through SA's amino groups by syntheticmethods known in the art. For example, Tgal-ATA in 0.1M HOAc pH 4.0 isneutralized by the addition of solid NaCO₃ (final concentration 0.1M, pH8.4) and a fifty fold molar excess is added to iodoacetylated SA. NH₂ OHis added to a final concentration of 0.05M and the solution mixedovernight at 37° C. under N₂. The resulting compound is SA covalentlymodified through SA's amino groups to contain trigalactose moieties(Tgal-SA). The synthesis of Tgal-SA is diagrammed in FIG. 14. Tgal-SAhas the formula ##STR4## Wherein n is 1 to 20.

In another example, Tgal-SH in 0.1 M HOAc is neutralized by the additionof NaCO₃ (final concentration 0.1M, pH 8) and a fifty fold molar excessis added to iodoacetylated SA and the solution mixed overnight at 37° C.after purging with N₂. Excess Tgal is removed, for example bycentrifugation using C30 CENTRICON filters and 0.05M PO₄, 0.15M NaCl pH7.5 buffer. The resulting compound is SA covalently modified through itsamino groups to contain trigalactose moieties.

The SA compounds containing covalently bonded carbohydrate moietiesmaintain the ability of SA to bind to biotin. For imaging or therapeuticmethods in which a modified biotin or biotin derivative or analog isemployed, the SA compounds of the invention may be derived from SAdesigned to bind to such a biotin species.

The ability of the SA compounds of the present invention to bind tobiotin can be determined by assays known to one of ordinary skill in theart, for example by a biotin binding saturation assay as describedhereinbelow.

For the trigalactose derivatives and modified SA compounds of thepresent invention, it is further contemplated to modify such compoundsby alkylation of the carbohydrate moieties. In particular, alkylationand preferably acetylation of galactose moieties is contemplated.Alkylation can be accomplished by synthetic methods known to one ofordinary skill in the art, such as disclosed for example by Lee (1982)Biochemistry 21:1045; Lee (1982) Biochemistry 21:6292, and Stowell(1980) Biochemistry 19:4904. Alkylation of carbohydrate moietiesprevents binding to the hepatic galactose receptor, and thus clearanceof the subject compounds can be slowed, if desired, until the alkylprotecting group is removed by serum esterases. Tomic et al (1991)Carbohydrate Res 210:191. Acetylated trigalactose derivatives such asTgal-ATA and Tgal-SH are particularly contemplated.

The Avid compounds of the present invention are provided bydeglycosylating and/or neutralizing Avid. The modified Avid compoundshave prolonged blood clearance relative to native Avid, and maintain theability of Avid to bind to biotin. Deglycosylated Avid (Avid-E) can beprovided by deglycosylating commercially available Avid by methods knownin the art, for example, by enzymatic digestion with endoglycosidase-H.For example, 5 mg/ml Avid in 0.05M sodium citrate, pH 5.5, is digestedwith 0.1 unit per ml endoglycosidase-H (Sigma, St. Louis, Mo.) for 18hours at 37° C. The extent of deglycosylation can be assessed byperforming SDS-PAGE and observing a decrease in apparent molecularweight of Avid due to removal of oligosaccharide. Deglycosylated avidinmay also be provided by recombinant methodology well-known to one ofordinary skill in the art.

Neutralized Avid (Avid-N) can be provided by alkylation of Avid's lysineresidues by methods known in the art. Alkylation by lower alkyl groups(C1-C10) is preferred. Acetylation is most preferred. For example,Avid's lysine amino acids may be neutralized by incubation with anN-hydroxysuccinimide ester of acetate (NHS-Ac). More specifically,acetic acid N-hydroxysuccinimide ester is incubated with avidin at molarratios of NHS-Ac/Avid ranging generally from 10 to 75. It is preferablethat the molar ratio of NHS-Ac/Avid be below 50. Neutralization can beassessed by monitoring a progressive decrease in isoelectric pointrelative to native Avid, for example, by polyacrylamide isoelectricfocusing.

In another aspect of the present embodiment, Avid is modified by bothdeglycosylation and neutralization of its lysine amino acids (Avid-E/N).A combination of deglycosylation and neutralization results insubstantially longer clearance times. Avid-E/N is conveniently preparedby deglycosylating and then neutralizing Avid as described hereinabove.

The appropriate degree of deglycosylation and/or neutralization is thatwhich results in prolonged blood clearance relative to native Avid whilemaintaining the ability of Avid to maintain to biotin, and furtherallows sufficient accumulation of a conjugated targeting agent at atarget site in the absence of unacceptable background levels as definedabove.

The modified Avid and SA compounds of the present invention maintain anadequate degree of the ability of native Avid and SA to bind to biotin.It is not necessary that the modified compounds exhibit the maximumtetrameric binding of Avid and SA for biotin. In a preferred embodiment,the present compounds exhibit at least about 25%, and more preferablyabout 50%, of the biotin saturation ratio of native Avid and SA asmeasured by an in vitro biotin binding saturation assay.

The ability of the compounds of the present invention to bind to biotincan be assessed by in vitro assays known to one of ordinary skill in theart. For example, the compounds of the present invention are incubatedwith a molar excess of radiolabeled biotin under conditions sufficientfor avidin-biotin or streptavidin-biotin binding. Biotin bound to thetested compound is separated from unbound biotin, for example byfiltration. The bound biotin/modified Avid or SA ratio is determined bydividing the activity in the retentate by the specific activity of theradiolabeled biotin. The binding ratios for each modified Avid or SAcompound are determined by dividing the bound moles of biotin by themoles of each compound.

In a preferred embodiment, the radiolabeled biotin is the biotin chelatedeferoxamineacetylcysteinylbiotin (DACB) labeled with ⁶⁷ Ga. DACB isknown in the art and described for example by Rosebrough et al (1993) J.Pharm. Exp. Therap. 265:408 and in U.S. Pat. No. 5,326,778. DACB can beradiolabeled by direct addition of carrier free ⁶⁷ Ga citrate (DuPontMerck Pharmaceutical Co., Billerica, Mass.). Samples of Avid, SA,modified Avid, and modified SA are incubated with a 20/1 molar excess of⁶⁷ Ga-DACB on CENTRICON-30 filters (Amicon, Beverly, Mass.) in 0.05MTris/0.15M NaCl, pH 7.5. After two washings, the bound DACB/proteinratio is determined by dividing the bound activity in the retentate bythe specific activity of ⁶⁷ Ga-DACB. The binding ratio for each compoundis determined by dividing the bound moles of DACB by the moles of eachcompound. Control Avid and SA generally exhibit a binding ratio of lessthan the maximum tetrameric binding of 4.0 in this assay. The modifiedAvid and SA compounds of the present invention exhibit binding ratios ofat least about 25%, and more preferably greater than about 50%, of thebinding ratio of control Avid and SA. The ordinarily skilled artisan canthus assess the compounds of the invention in the biotin binding assayto determine the appropriate amount of carbohydrate modification,deglycosylation and neutralization that is permissible without loss ofbiotin binding ability.

The blood clearance kinetics of the compounds of the present inventioncan be assessed by in vivo assays. The modified Avid and SA compoundsare detectably labeled, for example by radiolabeling, injected intoanimals, and blood clearance is then determined by biodistributionanalysis of radiolabeled samples. For example, the compounds of thepresent invention can be radiolabeled with ¹³¹ I by the Pierce lodobeadmethod (Pierce, Rodeford, Ill.) and separated from free iodine forexample by ultrafiltration using CENTRICON-30 filters. Experimentalanimals, for example, rabbits weighing about 3 kilograms, are fasted,anesthetized, and an external jugular vein is dissected and catheterizedfor injection and collection of blood samples. A radiolabeled compoundof the present invention is injected. Whole blood samples are taken atvarious time intervals for about two hours. By comparing the bloodclearance of galactose-modified SA with native SA, and Avid-E, theskilled artisan can determine the extent of modification that results inoptimal blood clearance.

The present invention further provides targeting agents conjugated tomodified SA and Avid. In a preferred embodiment the targeting agentbound to SA and Avid of the present invention is a protein or peptide.In a more preferred embodiment the targeting agent is a polyclonal ormonoclonal antibody or fragment thereof. Antibodies contemplated by thepresent invention include anti-tumor antibodies, anti-fibrin antibodies,anti-myosin antibodies and any lesion-specific antibodies. Anti-fibrinand anti-myosin antibodies are particularly useful in cardiac imaging.Non-specific IgG is also useful in accordance with the present inventionsince it can be used in targeting of abscesses via Fc receptor binding.More preferred targeting agents include humanized or chimericantibodies, human monoclonal or polyclonal antibodies, F_(v) fragments,Fab fragments, F(ab¹)2 fragments, single chain antibodies (SCA),molecular recognition units (MRU) and synthetic genetically engineeredbinding proteins or peptides. The most preferred targeting agents arefibrin- or fibrinogen-specific MAbs for the diagnosis of intravascularlesions (e.g. thrombi, emboli and atherosclerosis). Antifibrinmonoclonal antibody GC4 disclosed by Bini et al (1989) LaboratoryInvestigation 60:814 is particularly preferred. (ATCC No. ?).

Standard methods for the production and purification of antibodies andantibody fragments are known to one of ordinary skill in the art and canbe found, for example, in Antibodies: A Laboratory Manual, Harlow etal., eds, (1988) Cold Spring Harbor Laboratory.

Methods for conjugating the modified avidin or streptavidin of thepresent invention to targeting agents are known to one of ordinary skillin the art. Either or both of the targeting agent and the modified Avidor SA may be derivatized with a reactive group to facilitate bonding.For example, Ishikawa (1980) Immunoassay Supp. 1:1 and Duncan et al(1982) Anal. Biochem. disclose methods of sulthydryl-maleimide couplinguseful in the present invention. Kalofonos et al. (1990) J. Nucl. Med.31:1791 and Hnatowich et al. (1987) J. Nucl. Med. 28:1294 disclosemethods for conjugating an antibody with streptavidin and avidin,respectively, which use biotin as a linking group between the antibodyand streptavidin or avidin. Other synthetic methods are well-known toone of ordinary skill in the art.

A preferred method to conjugate the modified SA or Avid of the inventionto a monoclonal antibody comprises incorporating a sulfhydryl group intothe modified Avid or SA and a reactive maleimide residue into theantibody, and reacting these species to form a conjugate in which thefunction of the antibody and modified Avid or SA are retained. Aprotected sulfhydryl group can be incorporated byS-acetylthioacetylation of modified Avid or SA with SATA. A reactivemaleimide residue can be incorporated into an antibody for example byreaction of the antibody with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Alternatively, an antibody can bereacted with a variety of well-known bifunctional crosslinking agentsthat are reactive with a primary amine of the antibody and thesulfhydryl group of the modified Avid or SA.

Conjugates of modified SA and a targeting agent in which thecarbohydrate residues of SA are protected by alkylation as describedhereinabove are particularly contemplated.

The conjugates of modified Avid or SA and a monoclonal antibody or othertargeting agent are capable of binding to biotin or a biotin derivativeor analog, exhibiting clearance kinetics suitable to allow a timelysubsequent administration of biotin, and binding to a target in amountssuitable for diagnosis or therapy. Biotin binding and clearance kineticsmay be assessed as described hereinabove. The ability of the conjugateto bind to the target can be assessed by radiolabeling the conjugate,for example with ¹²⁵ I or ¹³¹ I as described hereinabove, injecting theconjugate into experimental animals as described hereinabove, and takingplanar gamma camera images at various time intervals for about twohours. At about two hours, animals are euthanized and major organs areremoved and biodistribution of the radiolabeled conjugate is analyzed bycounting organ samples in a gamma counter. By comparing the imagingprofile of the unmodified targeting agent with the conjugated targetingagent, it can be determined that the conjugated targeting agent hasmaintained specificity for the desired target.

Another aspect of the present invention is directed to a method ofdelivering a diagnostic or therapeutic agent to a target site. Themethod of delivery of a diagnostic agent comprises administering aconjugate of a modified Avid or SA of the present invention andtargeting agent to a host in an amount sufficient to bind to a targetsite, followed by administering a detectable biotinylated compound underconditions to form a complex with the target bound conjugate and at adose sufficient for detection. The resulting complex is then detected.The method of delivering a therapeutic agent to a target site comprisesadministering a conjugate of a modified Avid or SA of the presentinvention and a targeting agent to a host in an amount sufficient tobind to a target site, followed by administering a biotinylatedtherapeutic agent under conditions to form a complex with thetarget-bound conjugate and at a therapeutically effective dose.

The conjugates of the present invention have optimal clearance kineticsfor a method of diagnosis or therapy. The conjugates remain in thecirculation long enough for sufficient localization to a target site,but non-target bound conjugates are cleared rapidly enough to avoidundesirable background. Accordingly, in the method of the presentinvention the biotinylated compound may be administered within 24 hoursafter administration of the conjugate. In a preferred embodiment, thebiotinylated compound is administered within 6 hours, or more preferablywithin one hour, of administration of the conjugate.

A preferred embodiment of the present invention is a method of detectionof a thrombus or embolus comprising administering a conjugate of amodified Avid or SA and a fibrin-specific monoclonal antibody, followedby administering a detectable biotinylated compound, and detecting thecomplex of the biotinylated compound and target-bound conjugate. In apreferred embodiment the biotinylated compound is radiolabeled DACB, andthe SA is modified to contain trigalactose moieties.

The biotinylated compounds provide a delivery system for prodrugs,radioactive or paramagnetic metals, halogens, cytotoxins,chemotherapeutic drugs, fluorophores, enzymes or any other moiety thatcan be biotinylated.

Diverse classes of compounds, including proteins, polysaccharides,nucleic acids, haptens, peptides, chelating agents, halogenating agents,enzymes, fluorophores, lectins, cytotoxins, and drugs have beenbiotinylated in the prior art for a variety of applications. (See e.g.Diamondis et al. (1991) Clin. Chem. 37:625.)

Particularly preferred metal chelating agents include deferoxamine(DFO), diethylene-triaminopentaacetic acid (DTPA),ethylenediaminetetra-acetic acid (EDTA), bis-aminothiol (BAT, N₂ S₂),ethylenediamine-di(O-hydroxphenylacetic acid) (EDHPA), 2,2-dipyridyl(DIPY), polyaminopolycarboxylate, tetrazacyclododecane tetracetate(DOTA), dithiocarbamate, dithiosemicarbazone (DTS),tetraazacyclotetradecane-tetracetate (TETA), hydroxamic acid derivativesand prophyrins. In a preferred embodiment the metal chelating agent iscomplexed with a metal. In a most preferred embodiment the metal isTc-99m, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Bi, Fe, ⁵² Fe orGd.

Biotinylated compounds that contain a chelating agent are rendereddetectable or therapeutically effective by labeling with a radioactiveor paramagnetic metal. For radioimaging of a tissue or lesion ofinterest, it is preferred that the biotinylated compound is labeled withFe, Gd, ⁵² Fe, ⁶⁸ Ga, ⁹⁹ Tc, ¹¹¹ In, or ⁶⁷ Ga. For radioimmunotherapy,it is preferred that the DFO-biotin conjugate is labeled with ¹⁸⁶ Re,¹⁸⁸ Re, ²¹² Bi or ⁹⁰ Y. In radioimmunoimaging applications,administration of the labeled biotinylated compound is followed bydetection of the complex. The method used for diagnostic imaging isappropriate for the particular metal in the compound. For example,paramagnetic metal ions such as Fe and Gd are suitable for nuclearmagnetic resonance (NMR) analysis or magnetic resonance imaging (MRI).⁵² Fe and ⁶⁸ Ga are appropriate for analysis by positron emissiontomography (PET), whereas ^(99m) Tc, ¹¹¹ In and ⁶⁷ Ga can be detected bygamma camera imaging. The aforementioned means of image analysis areknown to one of ordinary skill in the art and can be conducted inaccordance with well-established techniques.

Similarly, the method of detection of other biotinylated compounds isdictated by the nature of the moiety that has been biotinylated.Detection is accomplished by methods known to one of ordinary skill inthe art.

In another preferred embodiment, the biotinylated moiety contains ahalogenating agent. Biotinylated compounds that contain a halogenatingagent may be rendered detectable or therapeutically effective bylabeling with a radioactive halogen. Preferred halogenating agentsinclude tyramine, aniline, Bolton Hunter reagent and stannane. In anespecially preferred embodiment the halogenating agent isradiohalogenated with an isotope of chlorine, bromine, iodine, fluorineor astatine. Preferred radioactive halogens include ²¹¹ At, ⁷⁷ Br, ¹²³I, ¹²⁵ I and ¹³¹ I. Methods for detecting halogens are known to one ofordinary skill in the art.

In another preferred embodiment the moiety to be biotinylated is afluorophore. Particularly preferred fluorophores are fluorescein,coumarin, rhodamine, phycoerythrin and Texas Red.

Preferred toxins include abrin, ricin, modeccin, Pseudomonas extoxin A,diphtheria toxin, pertussis toxin and Shiga toxin. Preferred enzymesinclude alkaline phosphatase, horseradish perioxidase, β-galactosidaseand glucose oxidase. Preferred therapeutic drugs include methotrexate,vinblastine, doxorubicin, bleomycin, cisplatimun, urokinase and tissueplasminogen activator.

In the method of delivery of the present invention, compounds can beadministered by well-known routes including oral, intravenous,intramuscular, intranasal, intradermal, subcutaneous, parenteral and thelike. Dosage of the conjugates agents of the present invention andbiotinylated compounds is an amount sufficient for the desiredtherapeutic or diagnostic effect, and can be determined by theordinarily skilled artisan guided by appropriate dosages for similarmethods of administration.

Another embodiment of the present invention provides a pharmaceuticalcomposition comprising a conjugate of a modified Avid or SA and atargeting agent together with a pharmaceutically acceptable carrier. Thecompounds can be administered by well-known routes including oral,intravenous, intramuscular, intranasal, intradermal, subcutaneous,parenteral and the like. Depending on the route of administration, thepharmaceutical compositions may require protective coatings.

The pharmaceutical forms suitable for injectionable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the ultimate solution form must be sterile andfluid. Typical carriers include a solvent or dispersion mediumcontaining, for example, water, buffered aqueous solutions (i.e.,biocompatible buffers), ethanol, polyol (glycerol, propylene glycol,polyethylene glycol and the like), suitable mixtures thereof,surfactants or vegetable oils. Sterilization can be accomplished by anyart recognized technique, including but not limited to, addition ofantibacterial or antifungal agents, for example, paraben, chlorobutanol,phenol, sorbic acid, thimerosal, and the like. Further, isotonic agents,such as sugars or sodium chloride may be incorporated in the subjectcompositions.

Production of sterile injectable solutions containing the subjectcontrast agent is accomplished by incorporating these agents in therequired amount in the appropriate solvent with various ingredientsenumerated above, as required, followed by sterilization, preferablyfilter sterilization. To obtain a sterile powder, the above solutionsare vacuum-dried or freeze-dried as necessary.

When the conjugates of the present invention are administered orally,the pharmaceutical composition thereof may also contain an inertdiluent, an assimilable edible carrier and the like, be in hard or softshell gelatin capsules, be compressed into tablets, or may be in anelixir, suspension, syrup or the like.

Preferred compositions of the conjugates provide diagnostically ortherapeutically effective dosages. Effective dosages can be determinedbased upon the particular conjugate, the route of administration, sizeand health of the patient, and so on.

As used herein, a pharmaceutically acceptable carrier includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic agents, and the like. The use of such media and agentsare well-known in the art.

The present invention is also directed to a kit for delivery of adiagnostic or therapeutic agent. In one embodiment, the kit iscompartmentalized to receive a first container adapted to contain amodified Avid or SA compound of the present invention conjugated to atargeting agent, and optionally a second container adapted to contain abiotinylated compound. In a preferred embodiment SA is modified tocontain trigalactose moieties and the targeting agent is a monoclonalantibody. In an exemplified use of the subject kit, the contents of thefirst container are administered to a host. A metal chelatingagent-containing biotinylated compound is labeled with a radioactive orparamagnetic metal and administered to the host within 24 hours afterthe administration of the targeting agent.

The following examples further illustrate the present invention.

EXAMPLE I

Synthesis and Characterization of SA-gal

This example provides the synthesis and characterization of modifiedstreptavidin containing covalently bonded galactose moieties. Galactosewas covalently bonded to SA via a nucleophilic reaction of the aminogroups of SA with α-D-galactopyranosyl-phenylisothiocyanate.

SA (International Enzymes, Inc., Fallbrook, Calif.) was solubilized in0.05 sodium phosphate/0.15 NaCl buffer, Ph 8.0 at a concentration of 10mg/ml as determined spectrophotometrically at 280nm (E₁,280nm^(1%)=34.0). α-Dgalactopyranosyl-phenylisothiocyanate (Sigma, St. Louis, Mo.)was solubilized in methanol and added to SA in molar incubation ratiosof 10, 25 and 50 for overnight incubation at room temperature. TheSA-galactose samples were then centrifuged using MICROCON-30 filters(Amicon, Beverly, Mass.).

Isoelectric focusing was performed to assess the conjugation ofgalactose to SA, since each conjugated galactose results in a decreaseof one positive charge. Polyacrylamide isoelectric focusing (IEF) wasperformed using a BioRad mini IEF cell (BioRad Labs, Hercules, Calif.)with ampholytes ranging from Ph 3-8. A progressive decrease in theisoelectric point of SA-gal samples was observed with increasing molarratios of galactose/SA, as shown in FIG. 1.

The molar ratio of bound galactose per SA was quantified by analyzingthe ultrafiltrate by an anthrone calorimetric assay as reported by Roe(1955) J.Biol. Chem. 212, 335-343!. For incubation ratios of 10, 25 and50 the average bound ratios were 5, 8 and 19 respectively. SA contains20 amino groups of which 16 are lysine amino and 4 are terminal aminoresidues. Thus, a galactose/SA molar ratio of 50 results in nearsaturation of SA with an average bound ratio of 19.

EXAMPLE II

Synthesis and Characterization of Avid-N, Avid-E and Avid-E/N

This example provides the synthesis of biochemically modified avidin,i.e. avidin neutralized by acetylation of its lysine amino acids(Avid-N), avidin deglycosylated by endoglycosidase-H (Avid-E), andavidin both neutralized and deglycosylated (Avid-E/N).

Avid (Sigma, St. Louis, Mo.) was neutralized by incubating acetic acidN-hydroxysuccinimide ester (NHS-Ac) (Sigma, St Louis, Mo.) with 5 mg/mlAvid in 0.05M sodium phosphate/0.15 NaCl buffer, Ph 8.0, at molar ratiosof 10, 25, 50 and 75 for one hour at room temperature to produce Avid-N.

Avid or Avid-N at a concentration of 5 mg/ml in 0.05 M sodium citrate,pH 5.5 buffer was digested with 0.1 unit per ml endoglycosidase-H(Sigma, St. Louis, Mo.) for 18 hours at 37° C. to produce Avid-E orAvid-E/N, respectively.

Avid-N and Avid-E were characterized by SDS-PAGE and IEF. SDS-PAGE wasperformed using a Bio-Rad mini-Proteam II cell (BioRad Labs, Hercules,Calif.) with 17% polyacrylamide gels. The results in FIG. 2B show thatthe subunits of Avid and Avid-N had equivalent molecular weights. Avid-Eexhibited a decrease in molecular weight due to the removal ofoligosaccharide. Polyacrylamide IEF was performed using a BioRad miniIEF cell (BioRad Labs, Hercules, Calif.) with ampholytes ranging from pH3-10. As shown in FIG. 2A, neutralization resulted in a progressivedecrease in isoelectric point, corresponding to the incubation ratio ofNHS-Ac/Avid. Deglycosylation had no effect on the IEF of Avid.

EXAMPLE III

Synthesis of Tqal-SA

The trigalactose derivative(6-aminohexamido)tris(B-D-galactosyloxymethyl)methane (T-gal) has beensynthesized and characterized by Lee (1978) Carbohydrate Research67:509. Tgal contains three galactose moieties at one end and a primaryamine at the other end. The trigalactose derivative of SA (Tgal-SA) wassynthesized as follows and as outlined in FIG. 3.

Synthesis of Tetra-O-acetyl-α-D-galactosyl bromide (Compound 1)

Acetic anhydride (100 ml) and 70% perchloric acid (0.6 ml) were mixed at5° C. and the solution was warmed to room temperature. D-galactose (10.0grams, 27.54 mmol) was added to the stirred mixture over five hours at arate that maintained the reaction temperature between 30°-40° C. Thereaction mixture was stirred further for 30 minutes at 35° C. Redphosphorous (3.0 grams, 96.9 mmol) was added and the flask cooled inice. Bromine (6.44 ml, 103.0 mmol) was added slowly to keep the reactiontemperature below 20° C., and 15.0 ml of water added at 20° C. over a 30minute period. The reaction mixture was stirred overnight at roomtemperature. Chloroform was added and the mixture filtered. The filtratewas washed with cold water three times and finally the organic layer waswashed with a saturated solution of sodium hydrogen carbonate. Thereaction mixture was dried over silicic acid, and chloroform wasevaporated under reduced pressure. The product was crystallized in ether(50ml) and hexane (100 ml) yielding 6.0 grams of product, melting point76°-77° C. (literature melting point 79°-84° C.). ¹ H NMR was inagreement with the structure of the compound.

Preparation of 6-(trifluoroacetamido)hexanoic acid (Compound 2)

6-Aminohexanoic acid (100 mmol, 13.1 g) was placed in a flask.Trifluoroacetic anhydride (270 mmol, 40.0 mL) was added to the flask atcold tap water temperature. After the addition of all trifluoroaceticanhydride, the reaction was brought to room temperature and stirred forone hour. The reaction mixture was evaporated at reduced pressure, 100ml of cold water was added and the mixture stirred overnight at 5° C.The crystalline product was filtered off, washed with cold water, driedand recrystallized from ether yielding 8.0 grams of product, meltingpoint 90°-91° C. (literature melting point 88°-89° C.). ¹ H NMR matchedthe structure of the compound.

Preparation of 2-(hydroxymethyl)-2-6(trifluoroacetamido)hexanamide!-1,3-propanediol (Compound 3)

A mixture of 2-amino-2-(hydroxymethyl)-1,3-propanediol (20 mmol, 2.42g), Compound 2 (25 mmol, 5.7g) and2-ethoxy-N-(ethoxycarbonyl)-1,2-dihydroquinoline (30 mmol, 7.4 g) wereboiled under reflux for 6 hours. The mixture was cooled to roomtemperature and evaporated to a syrup. Addition of ether yieldedcrystals. Recrystallization from ethyl acetate gave 4 grams of productwith melting point of 100°-101° C. (literature melting point 97°-99°C.). ¹ H NMR was in close agreement with the structure of the compound.

Preparation of (6-aminohexamido)tris (β-D-galactosyloxymethyl)methane(Compound 4)

A mixture of Compound 1 (7.29 mmol, 3.0 g), Compound 3 (1.45 mmol, 0.48g) and mercuric cyanide (7.3 mmol, 1.84 g) in 50 ml toluene:nitromethane(1:1, v/v) was stirred at 60° C. for 2 hours. Reaction mixture wasevaporated and was extracted with chloroform. The organic layer waswashed twice with 1M sodium chloride, dried and evaporated to a syrup.Silica gel TLC showed a single spot and therefore the compound was notpurified further. The galactose assay gave a weight:galactose ratio of1:3.

The resulting acetylated galactose compound was deprotected by stirringthe syrup overnight with 20 mM barium methoxide (25 ml) and evaporatingthe reaction mixture under reduced pressure. The reaction mixture wastreated with 1M sodium hydroxide (10 ml) and was stirred for 4 hours atroom temperature. The pH of the reaction mixture was lowered to 3.0 bythe addition of glacial acetic acid and was lyophilized. A portion (100mg) of this mixture was dissolved in water and was purified by a column(2×100 cm) of SEPHADEX G-10 in 0.1M acetic acid. The elution wasmonitored at 220 nm and the first peak was collected and lyophilized (5mg). The galactose assay of the product gave a weight:galactose ratio of1:3. Electrospray mass spectral analysis showed a quasimolecular ion atm/z 721 (M+H)⁺.

Preparation of SA-IA (Compound 5)

N-Hydroxysuccinimide iodoacetate (NHSIA, 1.17 mg) and SA (5 mg) weremixed in 50 mM phosphate buffer-saline containing 1 mM EDTA (pH 8.0).The reaction mixture was incubated for 1 hour at 37° C. and was washedwith 50 mM phosphate buffer-saline on a Centricon-30 filter.

Preparation of Tgal-ATA (Compound 6)

A 3/1 molar ratio of SATA in DMSO was added to 30μmoles of Tgal in 0.1MPO₄, pH 8.0, and incubated overnight at room temperature under N₂.Tgal-ATA was purified by G-10 SEPHEDEX size exclusion chromatography.

Preparation of SA-Tqal (Compound 7)

Tgal-ATA (Compound 6, 2.96 mg) and IA-SA (Compound 5, 2.0 mg) wereincubated overnight in the presence of N-hydroxylamine (0.5M) in 50 mMphosphate buffer-saline containing 1 mM EDTA (pH 8.4) at 37° C. Thebound Tgal-ATA:SA ratio was determined indirectly by the difference ingalactose concentration in the incubation mixture and that of the firstCENTRICON-30 filtrate a measured by the anthrone assay.

EXAMPLE IV

Biotin Binding Assay

This example demonstrates the ability of the modified avidin andstreptavidin compounds of Examples I-III to bind to biotin.

The biotin chelate deferoxamine acetyl cysteinylbiotin (DACB) wasprepared and purified as described by Rosebrough (1993) J. Pharm. Exp.Therap. 265:408 and U.S. Pat. No. 5,326,778.

The purified DACB was then used to assess the ability of the compoundsprepared in accordance with Examples I-III to bind to biotin. DACB wasradiolabeled by direct addition of carrier free ⁶⁷ Ga citrate (Du PontMerck Pharmaceutical Co., Billerica, Mass.). Separate samples of SA andAvid and modified SA and Avid prepared in accordance with ExamplesI-III, were incubated with a 20/1 molar excess of ⁶⁷ Ga DACB onCENTRICON-30 filters (Amicon, Beverly, Mass.) in 0.05 Tris/0.15M NaClbuffer, pH 7.5. After 2 washings, the bound DACB/protein ratio wasdetermined by dividing the bound activity in the retentate by thespecific activity of ⁶⁷ Ga DACB (˜2×10¹⁶ cpm/mole). The binding ratiosfor each sample were determined by dividing the bound moles of DACB bythe moles of each protein. As shown in FIG. 4A, control SA exhibited abinding ratio of 3.6, approximating maximum tetrameric binding. SA-galsamples with galactose/SA molar ratios of 10 and 25 exhibited noreduction in binding. The DACB saturation ratio for the SA-gal samplewith a galactose/SA molar ratio of 50 was 2.3. As shown in FIG. 4B, DACBsaturation analysis of Avid-N samples at molar ratios of 10 and 25approximated the binding of DACB to native avidin. Like the modified SAcompounds, the modified Avid compound did not exhibit a decrease inbiotin binding until large amounts of lysine neutralization hadoccurred.

Similarly, Tgal-SA prepared in Example IV exhibited binding to DACB tothe same extent as native SA.

EXAMPLE V

Biodistribution of Modified SA and Avid

SA (International Enzymes, Inc., Fallbrook, Calif.), Avid (Sigma, St.Louis, Mo.), SA-gal having average bound ratios of 5, 8 and 19 galactosemoieties per SA (SA-gal-5, SA-gal-8, and SA-gal-19, respectively),SA-Tgal, Avid-E, Avid-N (50 NHS-Ac/1 Avid) and Avid-E/N prepared inaccordance with Examples I-III were radiolabeled with ¹³¹ I by thePierce Iodobead method (Pierce, Rockford, Ill.) and free iodine removedby ultrafiltration using CENTRICON-30 filters. SA and Avid, 0.5 mg, wereradiolabeled with starting activities of ˜0.3mCi ¹³¹ I and ˜0.1mCi ¹³¹I, respectively. The radiolabeling yields were ˜75% for SA and ˜25% forAvid samples. When analyzed by Centricon-30 ultrafiltration, less than5% free iodine was present in the injected samples.

Biodistribution was determined in vivo. New Zealand White rabbits, ˜3kg, (n=3 per experiment) were fasted for 12 hours prior to theexperiment and anesthetized using an intramuscular injection ofchlorpromazine (25 mg/kg) followed by intravenous sodium pentobarbital(15 mg/kg). An external jugular vein was dissected and catheterizedusing a 5 French straight catheter for injection and collection of wholeblood. The animal was placed in a supine position under the gamma camerafor imaging and was then injected with 75 μg! of the radiolabeledproteins prepared above. Whole blood samples (2-3 ml) were taken atintervals for 2 hours. Planar gamma camera images (30K countacquisition) were taken at 5, 30, 60 and 120 minutes. At two hours,animals were euthanized. Major organs were removed, weighed andrepresentative samples taken for biodistribution analysis. Blood volumewas estimated to be 6% of total body weight.

FIG. 5A shows the blood clearance of samples of SA, SA-gal-5, SA-gal-8and SA-gal-19. SA and SA-gal-5 circulated similarly with greater than50% in the blood at 2 hrs. SA-gal-8 exhibited faster blood clearancewith ˜30% circulating at 2 hours, and SA-gal-19 exhibited the fastestclearance with 20% circulating at 2 hours. SA-Tgal, FIG. 9, shows thefastest clearance with approximately 4% circulating at 2 hours.Biodistribution results, FIG. 5B, indicate increasing hepatic uptake incorrelation with the amount of galactose bound per SA. This isconsistent with galactose receptor uptake by hepatocytes. Visual resultsfrom gamma camera images of rabbits injected with these samples were inagreement with the biodistribution results. ¹³¹ I-SA images, FIG. 6,show vascular activity as indicated by systemic circulation and cardiacactivity. ¹³¹ I-SA-gal samples showed increasing hepatic uptake anddecreased circulation with larger amounts of bound galactose. Hepaticmetabolism, as evident by biliary and gut activity, was also presentwith ¹³¹ I-SA-gal samples in the 30 and 120 minute images.

FIG. 7A shows the blood clearance of Avid, Avid-E, Avid-N and Avid-E/N.¹³¹ I-Avid cleared quickly from the circulation (FIG. 7A). ¹³¹ I-Avid-Eand ¹³¹ I-Avid-N cleared slower than native Avid, and the bloodclearance of ¹³¹ I-Avid E/N was substantially prolonged with circulatoryvalues at 2 hours approaching that of ¹³¹ I-SA. Thus, each modificationof Avid increased circulation time, with the slowest clearance resultingfrom a combination of deglycosylation and neutralization.Biodistribution results differed with each type of modification (FIG.7B). ¹³ I-Avid accumulated mostly in the liver and kidney. ¹³¹ I-Avid-Ekidney accumulation increased with a corresponding decrease in liveractivity. Conversely, ¹³¹ I-Avid-N kidney accumulation decreasedconsiderably but had a high liver accumulation. With ¹³¹ I-Avid-E/N,there were low levels in both the kidney and liver. These resultssuggest that the clearance of Avid consists of two mechanisms, one dueto net charge and one resulting from specific sugar receptor binding.

FIG. 8 shows planar gamma camera images of ¹³¹ I-Avid, ¹³¹ I-Avid-E, ³I-Avid-N and ¹³¹ I-Avid-E/N samples. Consistent with the biodistributionresults, ¹³¹ I-Avid activity was exclusively in the liver and kidneys.¹³¹ -Avid-E had increased kidney activity. ¹³¹ I-Avid-N images showed adramatic reduction in kidney activity and a corresponding increase inliver activity. ¹³¹ I-Avid-E/N showed circulatory activity with acorresponding decrease in liver and kidney activity. Urine activity waspresent with ¹³¹ I-Avid-E and ¹³¹ I-Avid-E/N and when analyzed by C-30centricon ultrafiltration, greater than 90% of the activity was proteinbound.

FIG. 10 shows planar gamma camera images of ¹³¹ I-Tgal-SA at 5, 30 and120 minutes. Consistent with the biodistribution results, ¹³¹ Tgal-SAimages demonstrated the liver as the single focus of tracer uptake withhepatic metabolism evident as indicated by gut activity in the 120minute image.

EXAMPLE VI

Synthesis of SA-GC4, gal-SA-GC4 and Tqal-SA-GC4

SA, SA-gal-19 and SA-Tgal were conjugated to GC4, an anti-fibrinmonoclonal antibody by the addition reaction of sulfhydryl-containing SAderivatives with maleimide-GC4 (GC4-mal) (as diagrammed in FIG. 11).

Preparation of GC4-mal (Compound 8)

A five fold molar excess of succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Pierce, Rockford,Ill.) solubilized in DMSO was added to a 16.9 mg/ml solution of GC4monoclonal antibody (Bini et al, 1989, Arteriosclerosis 9:109) in 0.05PO₄ /0.15M NaCl pH 7.5 buffer containing 1 mM EDTA for 1 hour. ExcessSMCC was removed by centrifugation using C 30 CENTRICON filters and 0.05PO₄ /0.15M NaCl pH 7.5 buffer containing 1 mM EDTA.

Preparation of Tgal-SH (Compound 9)

2-Iminothiolane (Traut's reagent) was added in two equal aliquots (t=0and 30 minutes) to Tgal, prepared according to Example III, for a finalmolar ratio of 20/1 in 0.1M PO₄ /1mM EDTA pH 7.6 and stirred for onehour at room temperature under N₂. The resulting Tgal-SH was purified byG-10 SEPHADEX (2.0×100 cm) size exclusion chromatography 0.1M aceticacid running buffer at a flow rate of 1.5ml/min.

Preparation of ATA-SA-gal (Compound 10)

SA-ATA was prepared by adding a six-fold molar excess of SATA to a 5mg/ml solution of SA in 0.05M PO₄ /0.15M NaCl pH 6.5 buffer containinglmM EDTA for two hours at room temperature. Excess SATA was removed bycentrifugation using CENTRICON-30 filters and 0.05M PO₄ /0.15M NaCl pH7.5 buffer containing 1mM EDTA.

A fifty-fold excess of α-D-galactopyranosylphenyl isothiocyanate (Sigma)solubilized in methanol was added to a 12 mg/ml solution of SA-ATA in0.05 PO₄ /0.5M NaCl pH 8 buffer overnight at room temperature. Excessgalactose was removed by centrifugation using C 30 CENTRICON filters and0.05 PO₄ /0.5M NaCl pH 7.5 buffer containing 1 mM EDTA.

Preparation of Tgal-SA-ATA (Compound 11)

IA-SA-ATA was synthesized by adding a 50 fold excess ofN-hydroxysuccinimide iodoacetamide (NHS-IA) in DMSO to a 5 mg/ml SA-ATAsolution 0.05 PO₄ /0.5M NaCl pH 8 buffer containing 1 mM EDTA andstirred overnight at 37° C. Excess NHS-IA was removed by centrifugationusing C 30 CENTRICON filters and 0.05 PO₄ /0.5M NaCl pH 7.5 buffer,containing 1 mM EDTA. A 50 fold excess of Tgal-SH (Compound 9) in 0.1MHoAC neutralized by the addition of NaCO₃ (final M=0.1M, pH=8), wasadded to IA-SA-ATA (final IA-SA-ATA concentration of 0.45 mg/ml)overnight at 37° C. after purging with N₂. Excess Tgal was removed bycentrifugation using C 30 CENTRICON filters and 0.05 PO₄ /0.5M NaCl pH 8buffer.

Preparation of gal-SA-GC4 (Compound 12)

Equal molar, 0.65 μM, gal-SA-ATA (Compound 10) and GC4-mal (Compound 8)were mixed in 0.05 PO₄ /0.15M NaCl pH 7.5 buffer containing 1 mM EDTAand 0.05M NH₂ OH overnight at room temperature. The gal-SA-GC4 conjugatewas purified from unreacted GC4, SA-gal and GC4-GC4 aggregates by sizeexclusion chromatography (SUPEROSE 12, Pharmacia, Piscataway, N.J.). ForSUPEROSE 12 chromatography, the running buffer was 0.05 PO₄ /0.15M NaClpH 9.0 at 0.3ml/min. For iminobiotin affinity chromatography, the secondpeak collected from the SUPEROSE 12 column was loaded with a 10 mlsuperloop onto a iminobiotin column. After the absorbance (A_(280mn))returned to baseline, the GC4-SA conjugates were eluted by changingbuffers to 0.1M sodium acetate pH 3.5. The eluent was then concentratedand the buffer exchanged by Centricon-30 centrifugation with 0.05 PO₄/0.15M NaCl pH 7.5 buffer. Each eluted protein peak was analyzed bySDS-polyacrylamide electrophoresis (to determine the extent ofcrosslinking) using a Bio Rad mini-Protean II cell (Bio Rad Labs,Hercules, Calif.) and 8.5% polyacrylamide gels.

Preparation of Tqal-SA-GC4 (Compound 13)

Equal molar, 0.65 μM, Tgal-SA-ATA (Compound 11) and GC4-mal (Compound 8)were mixed in 0.05 PO₄ /0.15M NaCl pH 7.5 buffer containing 1 mM EDTAand 0.05 M NH₂ OH overnight at room temperature. The Tgal-SA-GC4conjugate was purified from unreacted GC4, SA-Tgal and GC4-GC4aggregates by size exclusion chromatography (SUPEROSE 12, Pharmacia,Piscataway, N.J.). For SUPEROSE 12 chromatography, the running bufferwas 0.05 PO₄ /0.15M NaCl pH 9.0 at 0.3 ml/min. For iminobiotin affinitychromatography, the second peak collected from the SUPEROSE 12 (highlycrossed-linked beaded agarose Suspension in 20% ethanol; Fractionationrange: (MW) globular protein 1,000-300,000; wet bead diameter: 20-40 uM)column was loaded with a 10 ml superloop onto a iminobiotin column.After the absorbance (A_(280mn)) returned to baseline, the GC4-SAconjugates were eluted by changing buffers to 0.1M sodium acetate pH3.5. The eluent was then concentrated and the buffer exchanged byCENTRICON-30 centrifugation with 0.05 PO₄ /0.15M NaCl pH 7.5 buffer.Each eluted protein peak was analyzed by SDS-polyacrylamideelectrophoresis (to determine the extent of crosslinking) using a BioRad mini-Protean II cell (Bio Rad Labs, Hercules, Calif.) and 8.5%polyacrylamide gels.

Preparation of SA-GC4 (Compound 14)

Equal molar, 0.65 μM, SA-ATA (Compound 5, Example IIB) and GC4-mal(Compound 8) were mixed in 0.05 PO₄ /0.15M NaCl pH 7.5 buffer containing1 mM EDTA and 0.05M NH₂ OH overnight at room temperature. The SA-GC4conjugate was purified from unreacted GC4, SA and GC4-GC4 aggregates bysize exclusion chromatography (SUPEROSE 12, Pharmacia, Piscataway,N.J.). For SUPEROSE 12 chromatography, the running buffer was 0.05 PO₄/0.15M NaCl pH 9.0 at 0.3 ml/min. For iminobiotin affinitychromatography, the second peak collected from the SUPEROSE 12 columnwas loaded with a 10 ml superloop onto a iminobiotin column. After theabsorbance (A_(280mn)) returned to baseline, the GC4-SA conjugates wereeluted by changing buffers to 0.1M sodium acetate pH 3.5. The eluent wasthen concentrated and the buffer exchanged by CENTRICON-30centrifugation with 0.05 PO₄ /0.15M NaCl pH 7.5 buffer. The elutedprotein peak was analyzed by SDS-polyacrylamide electrophoresis (todetermine the extent of crosslinking) using a Bio Rad mini-Protean IIcell (Bio Rad Labs, Hercules, Calif.) and 8.5% polyacrylamide gels. Theresults are shown in FIG. 12. In the presence of SDS, SA dissociatesinto its 15 kdal subunits. Therefore, GC4-SA conjugates are evident as15 kdal band increments heavier than 150 kdal GC4.

EXAMPLE VII

This example provides the method to investigate the coupling of theprimary amines of SA with N-hydroxysuccinimide.

N-Succinimidyl-3- 2-pyridyldithio!propionate (SPDP) (Pierce, Rockford,Ill.) solubilized in methanol was added to 5 mg/ml SA (InternationalEnzymes, Fallbrook, Calif.) solution in molar SPDP/SA ratios rangingfrom 2-50 in 0.05 PO₄ /0.15M NaCl pH 6.5 and pH 8.0. After a two hourincubation, free SPDP was removed from the bound adduct (PDP) by fourwashings on CENTRICON-30 filters (Amicon, Beverly, Mass.). For eachsample, bound PDP was quantified spectrophotometrically by the releaseof pyridine-2-thione in the presence of 1 mM DTT (E₁,343nm^(1M)=8.08×10³ M). SA concentrations were also determinedspectrophotometrically (E=34) by the release of release ofpyridine-2-thione after disulfide bound cleavage with DTT. FIG. 13Agraphs the bound ratio of PDP/SA compared to the SPDP/SA incubationratio at pH 6.5 and 8.0. The maximum bound ratio approached 20, which isconsistent with the total number of amines per mole of SA (16 lysineamino acids and 4 terminal amines).

Isoelectric focusing analysis on these samples, FIG. 13B, showed adecrease in pI associated with the degree of neutralization of the SAamines during PDP crosslinking.

SA was incubated with SATA (Compound 5, Example III) at a 6/1 SATA/SAmolar ratio at pH 6.5 which gave an average PDP/SA ratio of 2. Toconfirm the extent of ATA crosslinking, hydroxylamine was added toATA-SA, and the concentration of SH determined with Ellman's reagent.The resultant SH/SA ratio was 2.2.

Bound gal/SA-ATA (Compound 10, Example VII) and Tgal/SA-ATA (Compound11, Example VI) ratios were 18 and 13 respectively as assayed by ananthrone calorimetric assay as reported by Roe (1955) J.Biol. Chem. 212,335-343!. For SA-Tgal, this is equivalent to an absolute galactose/SAratio of 39.

The four terminal α-amines of SA are more reactive than the ε-amines dueto their lower pKa and are thus preferred for cross-linking. Theterminal amines are appropriate for cross-linking to monoclonalantibodies since the N-terminal domains are flexible and not involved inbiotin binding or subunit association. Pahler et al (1987) J. Biol.Chem. 261:13911; Hendrickson et al (1989) Proc. Natl. Acad. Sci.86:2190.

EXAMPLE VIII

Radiolabeling, Biodistribution and Imaging GC4-SA, (Compound 14, ExampleVI), gal-SA-GC4, (Compound 12, Example VI) and Tgal-SA-GC4, (Compound13, Example VI) were radiolabeled with ¹³¹ I or ¹²⁵ I by the PierceIodobead method (Pierce, Rockford, Ill.) and free iodine removed byultrafiltration using CENTRICON-30 filters.

Biodistribution was determined in vivo. New Zealand White rabbits, ˜3kg, were fasted for 12 hours prior to experiment and anesthetized usingan intramuscular injection of chlorpromazine (25 mg/kg) followed byintravenous sodium pentobarbital (15 mg/kg). An external jugular veinwas dissected and catheterized using a 5 French straight catheter forinjection and collection of whole blood. The animal was placed in asupine position under the gamma camera for imaging and was then injectedwith ˜75 μg (˜30 μCi) of the radiolabeled proteins prepared above. Wholeblood samples (2-3ml) were taken at intervals for 2 hours. Planar gammacamera images (30K count acquisition) were taken at 5, 30, 60 and 120minutes. At two hours, animals were euthanized. Major organs wereremoved, weighed and representative samples taken for biodistributionanalysis. Blood volume was estimated to be 6% of total body weight. FIG.14A graphs the blood clearance of ¹²⁵ I-SA-GC4, ¹³¹ I-gal-SA-GC4 and ¹³¹I-Tgal-SA-GC4. ¹²⁵ I-SA-GC4 had the slowest blood clearance, withapproximately 87% circulating at 120 minutes. ¹³¹ gal-SA-GC4 had fasterclearance with ˜59% circulating at 120 minutes and ¹³¹ Tgal-SA-GC4 hadthe fastest clearance with ˜21 and 11% circulating at 10 and 120minutes, respectively.

Biodistribution results, FIG. 14B, show a moderate increase in liveraccumulation of ¹³¹ I-gal-SA-GC4 and ¹³¹ I-Tgal-SA-GC4 and minimalaccumulation in other organs.

FIG. 15 is a composite of the nuclear images of ¹³¹ I-gal-SA-GC4 and ¹³¹I-Tgal-SA-GC4 at 5, 30 and 120 minutes after injection into the rabbit.¹³¹ I-gal-SA-GC4 images are consistent with the pharmacokinetic results,as shown by heart, lung and blood activity, compared to the faster bloodclearance and immediate liver accumulation of ¹³¹ I-Tgal-SA-GC4.

I claim:
 1. A conjugate of a targeting agent and a covalently modifiedstreptavidin, wherein said covalently modified streptavidin contains atleast one carbohydrate moiety, said carbohydrate moiety being galactose,mannose, fructose or lactose which is covalently attached to thestreptavidin.
 2. The conjugate of claim 1 wherein said targeting agentis a monoclonal antibody.
 3. The conjugate of claim 2 wherein saidmonoclonal antibody is an anti-fibrin monoclonal antibody.
 4. Aconjugate of a trigalactose modified streptavidin and a monoclonalantibody, said trigalactose-modified streptavidin (SA) having theformula ##STR5## wherein n is 1 to 20 and gal is galactosyl.
 5. Apharmaceutical composition comprising the conjugate of claim 1 or 4 anda pharmaceutically acceptable carrier.
 6. A method of delivering adiagnostic agent to a target site comprising administering the conjugateof claim 1 or 4 to a host in an amount sufficient to bind to a targetsite; administering a detectable biotinylated compound under conditionssufficient to form a complex with target bound conjugate and at a dosesufficient for detection; and detecting said complex.
 7. A method ofdelivering a therapeutic agent to a target site comprising administeringthe conjugate of any one of claim 1 or 4 to a host in an amountsufficient to bind a target site; and administering a therapeuticallyeffective amount of a biotinylated compound under conditions sufficientto form a complex with target bound conjugate.
 8. A compartmentalizedkit for delivery of a diagnostic or therapeutic agent wherein said kitcomprises a first container adapted to contain the conjugate of any oneof claim 1 or
 4. 9. The kit of claim 8 further comprising a secondcontainer adapted to contain a biotinylated compound.
 10. The conjugateof claim 1 wherein said carbohydrate moiety is a galactose moiety. 11.The conjugate of claim 10 wherein said galactose moiety is covalentlybonded to streptavidin through an amino group of streptavidin.
 12. Theconjugate of claim 10 wherein said galactose moiety is trigalactose. 13.The conjugate of claim 11 containing about 1 to about 20 galactosemoieties.
 14. The conjugate of claim 12 containing about 1 to about 20trigalactose moieties.
 15. The conjugate of claim 1 wherein thecovalently modified streptavidin is trigalactose modified steptatvidinof the formula ##STR6## wherein n is 1 to 20 and gal is ##STR7##